CN110144341B - Alginate lyase mutant - Google Patents

Alginate lyase mutant Download PDF

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CN110144341B
CN110144341B CN201810143677.9A CN201810143677A CN110144341B CN 110144341 B CN110144341 B CN 110144341B CN 201810143677 A CN201810143677 A CN 201810143677A CN 110144341 B CN110144341 B CN 110144341B
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CN110144341A (en
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许丽红
石增秀
周利伟
黄亦钧
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Weifang Kdn Biotech Co ltd
Qingdao Vland Biotech Group Co Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02003—Poly(beta-D-mannuronate) lyase (4.2.2.3)

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Abstract

The invention relates to the technical field of protein engineering modification, and in particular relates to an alginate lyase mutant. The expression level of the alginate lyase mutant obtained by screening through a random mutation method in the bacillus subtilis is obviously improved. The activity of the shake flask fermentation supernatant of the bacillus subtilis for recombinant expression of the wild type alginate lyase is 488U/ml, while the shake flask fermentation supernatant of the bacillus subtilis for recombinant expression of the alginate lyase mutant is up to 635-780U/ml, which is improved by 30-60 percent, and unexpected technical effects are achieved. The application of the mutant is favorable for reducing the production cost of the alginate lyase, and the market prospect is wide.

Description

Alginate lyase mutant
Technical Field
The invention relates to the technical field of protein engineering modification, and particularly relates to an alginate lyase mutant and application thereof.
Background
Alginic acid is a marine polysaccharide second in reserve to cellulose. Alginic acid is present in the cell walls and intercellular substance of seaweeds, and is most abundant in brown algae, and most giant brown algae are potential sources of alginic acid. However, different kinds of brown algae contain different alginic acids, so the source of brown algae is selected according to the availability of brown algae and the nature of alginic acid. The main commercial sources of alginic acid are ascophyllum nodosum, bull alga, pterocyta, kelp, gulfweed, and hornworts, of which kelp, and ascophyllum nodosum are the most important. The world's origin of alginic acid is mainly distributed in coastal countries and regions such as norway, usa, france, china, japan and korea.
At present, methods for degradation of alginic acid can be divided into three major categories: one is a chemical degradation method, and an alkali hydrolysis method is widely adopted at present, which has the defects of environmental pollution and large energy consumption, and the alkali hydrolysis damages effective substances in the seaweed. In addition, there is a hydrogen peroxide oxidation degradation method; the second is physical degradation, such as sonication of alginic acid; the third type is an alginate lyase enzymolysis method, the conditions for degrading alginic acid by an enzyme method are mild, the process is controllable, the yield is high, the method is green and safe, the environment is friendly, the action mechanism is clear, the product is determined, and enzyme preparations with different substrate specificities can be singly or combined according to the specific target product requirements.
The alginate lyase cleaves 4-O-glycosyl bonds of alginic acid through beta elimination reaction, and simultaneously forms double bonds between C-4 and C-5, so that 4-deoxy-L-erythro-hex-4-ene pyranosyluronate with strong absorption peak at 230-240mm is generated at the non-reducing end of the generated oligosaccharide. Alginate lyase is classified into three types according to the specificity of a substrate: the first is a polymannuronate lyase, specific for polyM; the second is polyguluronate lyase, specific for polyG; the third is a lyase specific for polyMG. Can be divided into endo-alginate lyase and exo-alginate lyase according to the action mode.
The alginate lyase has wide sources, and mainly has three main types, wherein the first type is microorganisms, such as marine bacteria, soil bacteria, fungi and the like; the second kind is marine mollusk and echinoderm, such as conch, stichopus japonicus, abalone, etc.; the third is plants such as Macrocystis, ascophyllum nodosum, and Laminaria japonica.
At present, most alginate lyase is produced by using alginate-decomposing bacteria. Although the wild type alginic acid decomposition bacteria can effectively obtain quantitative enzyme protein, the yield is very low, the cost is high, and the practical application requirements are difficult to achieve. Therefore, the heterologous expression of the alginate lyase gene by using a genetic engineering means is the most effective way for improving the yield of the alginate lyase. The research mainly focuses on the cloning of alginate lyase genes of the alginate decomposing bacteria and the overexpression in escherichia coli. At present, more than twenty alginate lyase genes of alginate-decomposing bacteria have been cloned, and most of them have been successfully expressed heterologously. The expression quantity of the recombinant alginate lyase is higher than that of the wild strain. In 1993, researchers such as Maki et al expressed the alginate lyase from Pseudomonas sp. OS-AIG-9 in E.coli, with 53-fold higher recombinase activity than the wild strain.
In recent years, with the wide application of protein engineering technology in the field of enzyme preparations, the development of novel alginate lyase with high enzyme activity level and excellent property becomes a research hotspot in the field, and has important significance for reducing the production cost of the alginate lyase and promoting the industrialization of the alginate lyase.
Disclosure of Invention
The invention provides a novel alginate lyase mutant for solving the problems in the prior art, and a bacillus subtilis engineering strain for recombining and expressing the mutant is constructed. Compared with wild alginate lyase, the expression level of the alginate lyase mutant in bacillus subtilis is obviously improved, the production cost of the alginate lyase is greatly reduced, and the market prospect is wide.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an alginate lyase mutant, which has any one of amino acid sequences shown in (I), (II) or (III):
(I) a sequence having at least 95% homology with the amino acid sequence SEQ ID NO 1 of alginate lyase;
(II) has at least one immune epitope of the alginate lyase in the (I), and the amino acid sequence of the alginate lyase is obtained by modifying, substituting, deleting or adding one or more amino acids;
(III) an amino acid sequence encoded by a nucleotide sequence shown by SEQ ID NO:2 or a complementary sequence thereof or a sequence that differs from the nucleotide sequence shown by SEQ ID NO:2 or a complementary sequence thereof due to degeneracy of the genetic code;
in other embodiments of the invention, the substitution is a substitution of 1 amino acid.
In other embodiments of the invention, the substitution comprises a substitution at any one of amino acids 33, 49, 50, 53, 79, 133, 188, 196, 203, 205, 239, 243, 271 of the alginate lyase having the amino acid sequence of SEQ ID NO. 1.
In other embodiments of the invention, the substitution comprises changing amino acid 33 from K to N.
In other embodiments of the invention, the substitution comprises changing amino acid 49 from D to N.
In other embodiments of the invention, the substitution comprises changing the amino acid at position 50 from K to G.
In other embodiments of the invention, the substitution comprises changing amino acid 53 from K to R.
In other embodiments of the invention, the substitution comprises changing amino acid 79 from P to D.
In other embodiments of the invention, the substitution comprises changing amino acid 133 from D to N.
In other embodiments of the invention, the substitution comprises changing amino acid 188 from K to D.
In other embodiments of the invention, the substitution comprises changing amino acid 196 from P to S.
In other embodiments of the invention, the substitution comprises changing amino acid 203 from E to S.
In other embodiments of the invention, the substitution comprises a change of amino acid 205 from a to S.
In other embodiments of the invention, the substitution comprises changing amino acid 239 from L to I.
In other embodiments of the invention, the substitution comprises changing amino acid 243 from E to K.
In other embodiments of the invention, the substitution comprises changing amino acid 271 from K to N.
In other embodiments of the invention, the amino acid sequence of the alginate lyase mutant is as shown in SEQ ID NO.
The invention also provides a DNA molecule encoding the amino acid sequence.
The invention also provides a recombinant expression vector with the DNA molecule.
The invention also provides a bacillus subtilis (B.subtilis)Bacillus subtilis) Comprising the above recombinant expression vector.
The invention also provides application of the bacillus subtilis in producing alginate lyase.
Compared with wild alginate lyase, the expression level of the alginate lyase mutant obtained by screening through a random mutation method in the invention in bacillus subtilis is obviously improved. The enzyme activity of the shake flask fermentation supernatant of the bacillus subtilis for recombinant expression of the wild type alginate lyase is 488U/ml, while the shake flask fermentation supernatant of the bacillus subtilis for recombinant expression of the alginate lyase mutant is up to 635-780U/ml, which is improved by 30-60%, and unexpected technical effects are achieved. The efficient expression of the mutant in the bacillus subtilis is beneficial to reducing the production cost of the alginate lyase, and the market prospect is wide.
Detailed Description
The present invention uses conventional techniques and methods used IN the fields of genetic engineering and MOLECULAR BIOLOGY, such as those described IN MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the field on the basis of the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.
In the embodiment of the invention, the culture medium comprises the following components in percentage by weight:
LB plate: tryptone 1%, yeast powder 0.5%, naCl 1%, agar 1.5%;
LB liquid medium: tryptone 1%, yeast powder 0.5%, naCl 1%;
1 minimum salt solution: k 2 HPO 4 14 g/L，KH 2 PO 4 6 g/L，（NH 4 ） 2 SO 4 2 g/L, trisodium citrate 1 g/L, mgSO 4 •7H 2 O 0.2 g/L；
GM I solution: 95.6 ml of minimum salt solution, 2.5 ml of 20% glucose, 0.4 ml of 5% hydrolyzed casein, and 10% yeast powder juice 1 ml;
GM II solution: 96.98 ml of minimum salt solution, 2.5 ml of 20% glucose, 0.08 ml of 5% hydrolyzed casein, 0.04 ml of 10% yeast powder juice, 1M MgCl 2 0.25 ml，1 M CaCl 2 0.05 ml；
Beef extract peptone medium: beef extract 0.5%, peptone 1%, naCl 0.5%, agar 1.5%, pH 7.2.
Example 1 screening of alginate lyase mutants
In order to improve the enzyme activity of wild-type alginate lyase AL (the amino acid sequence is SEQ ID NO:1, the coding nucleotide sequence is SEQ ID NO:2, synthesized by Shanghai Biotechnology, inc.), the applicant screened a large number of mutations by directed evolution technology, and designed PCR primers AL-F1 and AL-R1 as follows:
AL-F1：ggcgttcagcaacatgagcgcgcaggctcaggataaaaaatctaaatct;
AL-R1：ccgtcctctgttaacctcgagttattattaatgcgtcacctgaagacta；
taking a wild type alginic acid lyase AL gene SEQ ID NO:2 as a template, carrying out PCR amplification by using the primer and a GeneMorph II random mutation PCR kit (Stratagene), recovering a PCR product by using a gel, connecting the PCR product to pSZX302 plasmid, transforming into escherichia coli BL21 (DE 3), coating the escherichia coli BL21 (DE 3) on an LB + Amp flat plate, carrying out inversion culture at 37 ℃, after transformants appear, picking the transformants to a 96-well plate one by using toothpicks, adding 150 mu L of LB + Amp culture medium containing 0.1 mM G into each well, culturing at 37 ℃, culturing at 220rpm for about 6 h, centrifuging, discarding supernatant, carrying out resuspension on the thalli by using a buffer solution, and repeatedly breaking the walls to obtain the escherichia coli cell lysate containing the alginic acid IPT lyase.
50 mu L of lysate is respectively taken to two new 96-well plates, and the alginate lyase enzyme activity is respectively measured under the condition of pH7.5. As a result, compared with the wild type alginate lyase AL, the enzyme activity of some mutants under the condition of pH7.5 is not changed, and the enzyme activity of some mutants is even reduced. Finally, the applicant screened for a mutation site that significantly increased the alginate lyase activity at ph 7.5: K33N, D49N, K50G, K53R, P79D, D133N, K188D, P196S, E203S, a205S, L239I, E243K and K271N.
The alginate lyase mutant containing K33N single-point mutation is named as ALm1, and the amino acid sequence of the mutant is SEQ ID NO:3;
the alginate lyase mutant containing the D49N single-point mutation is named as ALm2, and the amino acid sequence of the mutant is SEQ ID NO:4;
the alginate lyase mutant containing K50G single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:5;
the alginate lyase mutant containing K53R single-point mutation is named as AL-m4, and the amino acid sequence is SEQ ID NO:6;
the alginate lyase mutant containing P79D single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:7;
the alginate lyase mutant containing D133N single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:8;
the alginate lyase mutant containing the K188D single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:9;
the alginate lyase mutant containing P196S single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:10;
the alginate lyase mutant containing the E203S single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:11;
the alginate lyase mutant containing A205S single-point mutation is named as ALm10, and the amino acid sequence of the mutant is SEQ ID NO:12;
the alginate lyase mutant containing L239I single-point mutation is named as ALm, and the amino acid sequence of the mutant is SEQ ID NO:13;
the alginate lyase mutant containing E243K single-point mutation is named as ALm12, and the amino acid sequence of the mutant is SEQ ID NO:14;
the alginic acid lyase mutant containing the K271N single-point mutation is named as ALm, and the amino acid sequence of the alginic acid lyase mutant is SEQ ID NO:15.
the 13 alginate lyase mutants were synthesized by Shanghai Czeri Bio Inc. as the coding gene fragments.
And respectively connecting the synthesized alginate lyase mutant gene fragments to a pSZX302 plasmid to obtain recombinant plasmids, and sending the recombinant plasmids to a Beijing Huada gene research center for sequencing analysis. The sequencing results demonstrated that no amplification errors occurred.
And (3) purifying the plasmid from the Escherichia coli clone with correct sequencing result by using a plasmid intermediate quantity preparation kit (Axygen) to obtain the recombinant expression plasmid carrying the alginate lyase mutant gene sequence.
The gene segment of the wild alginate lyase AL is obtained by amplification by the same method, and the recombinant expression plasmid carrying the gene sequence of the wild alginate lyase AL is constructed.
Example 2 transformation and screening
2.1 transformation
The recombinant expression plasmids are respectively transformed into host bacteria bacillus subtilis F4 (Bacillus subtilisF4 Respectively obtaining the bacillus subtilis recombinant strains for recombinant expression of wild-type alginate lyase AL and alginate lyase mutant ALm, ALm, … … and ALm.
The specific transformation process is as follows: inoculating the freshly activated bacillus subtilis F4 into 5 ml of GM I solution by an LB plate, and carrying out shaking culture at 30 ℃ and 125 rpm overnight to obtain a culture solution A; transferring 2ml culture solution A into 18 ml GM I solution, culturing at 37 deg.C and 250 rpm for 3.5 h to obtain culture solution B; transferring 10 ml culture solution B into 90 ml GM II solution, culturing at 37 deg.C and 125 rpm for 90 min to obtain culture solution C;5000 And g, centrifugally collecting the thalli in the culture solution C for 10min, and lightly suspending the thalli by using 10 ml of GM II solution, wherein the suspended thalli are competent cells. Then, an appropriate amount of alginate lyase DNA amplified in example 1 was added to 0.5 mL competent cells, cultured with shaking at 37 ℃ and 200 rpm for 30 min, then coated on an LB plate (containing 5 μ g/mL chloramphenicol), cultured overnight at 37 ℃, and the transformants were checked and verified the next day.
Shake flask fermentation screening
The Bacillus subtilis recombinant strains described in example 2.1 were inoculated into 50mL of liquid fermentation medium (yeast extract 0.5%, tryptone 0.5%, glucose 1%, K) 2 HPO 4 1.8 percent) and 48 h by shaking flask fermentation, centrifuging for 5000 g and 10min, collecting supernatant, and respectively measuring the activity of alginic acid lyase in the supernatant.
The method for measuring the enzyme activity of the alginate lyase comprises the following steps:
1. definition of enzyme Activity Unit
Under the conditions of 40 ℃ and pH7.5, in the reaction system specified in the method, the substrate sodium alginate is degraded every minute to generate unsaturated bonds, and the increase of absorbance by 0.1 at 235nm is one enzyme activity unit U.
2. Principle of
The alginate lyase can cut off glycosidic bonds in alginate molecules through beta-elimination reaction to generate unsaturated double bonds at non-reducing ends, and the double bonds are positioned at non-reducing tail ends C of products 4 、C 5 And produces a maximum uv absorption at 235 nm.
3. Measurement method
3.1 Liquid sample: diluting with buffer solution to appropriate times, and controlling the absorbance value OD 235 Between 0.22 and 0.35, the enzyme activity is about 0.5U/mL.
3.2 Measurement procedure
Enzyme reaction: taking three test tubes (15mm. Times. 150mm), adding 1.8mL of substrate, preheating in 40 ℃ water bath for 5min, adding 0.2mL of diluted enzyme solution, accurately timing, carrying out vortex oscillation, keeping the temperature at 40 ℃ for 10min, taking the test tubes out of the water bath, immediately adding 2mL of phosphoric acid stop solution, carrying out vortex oscillation, and placing the test tubes on a test tube rack outside a water bath kettle.
Blank: adding 1.8mL of substrate into a test tube with the thickness of 15mm × 150mm, preheating in a water bath at 40 ℃ for 5min, adding 0.2mL of buffer solution, carrying out vortex oscillation, preserving the temperature at 40 ℃ for 10min, taking out the test tube from the water bath, immediately adding 2mL of phosphoric acid stop solution, carrying out vortex oscillation, and placing the test tube on a test tube rack outside a water bath kettle.
Color comparison: immediately after the blank and the enzyme reaction of each sample had terminated, the color was taken at 235nm and the absorbance A was recorded 0 And A Sample (A) 。
Remarking:
8. computing
X=（A 0 －A Sample (A) ）×2×N/（t×0.1）
In the formula:
x-enzyme activity, U/mL or U/g
2-volume factor of 2mL of phosphoric acid stop solution
t (min) -enzymatic reaction time (in the linear range of the enzymatic reaction)
0.1-system coefficient, i.e. conversion of the absorbance increase unit to 0.1
N-dilution multiple
Through simplification: enzyme activity (U/mL) = (A) 0 －A Sample (II) ）×2×N。
The present invention will be described in detail with reference to specific embodiments.
The enzyme activity determination result shows that the enzyme activity of the fermentation supernatant of the bacillus subtilis for recombining and expressing wild type alginate lyase is 488U/ml, while the enzyme activity of the bacillus subtilis for recombining and expressing alginate lyase mutants is up to 635-780U/ml, which is improved by 30-60%. Therefore, the expression level of the alginate lyase mutant ALm, ALm, … … and ALm in bacillus subtilis is obviously higher than that of the wild-type alginate lyase AL, and unexpected technical effects are achieved.
Sequence listing
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WEIFANG KDN BIOTECH Co.,Ltd.
<120> alginate lyase mutants
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Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
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100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
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Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
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Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
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Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
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gtcacggtcc ctgaagaaaa tccggataaa ccgggcaaac cgtatagctt aggctatccg 180
gaaatcttga attatgcgga agataaaatt gcgagtaaat atatgtatga tgatccgaaa 240
gataaatcag ttgtctttta tgcgtttccg agcggcgtga cgacagcgaa tacgcattat 300
agccgctcag aacttcgcga aacaatggaa acaggaagca ataaagttaa ttggacattt 360
gctaaaggag gcaaaatgag aggcacgtat gcgatcgatg atattagcaa agaaccggat 420
ggcaaatatt ctcgggttat catcgcgcag attcatggag tgttgacgga tgaacaacgt 480
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ggcaaaattc gcgtcaaaac gaaagtgctg aaagacctta atgcgccgta taaagaaatg 600
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tattttaaag cgggcaatta ttttcagagc aaaacacctg gcacgtttgc taaagtcaaa 840
atctattcac ttcaggtgac acat 864
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Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
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Asn Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
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Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
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Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
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20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asn Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 5
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Gly Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 6
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Arg Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 7
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Asp Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 8
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asn Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 9
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Asp Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 10
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Ser Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 11
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Ser His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 12
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ser Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 13
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Ile Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 14
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Lys Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Lys Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285
<210> 15
<211> 288
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Ser Ile Gln Phe Ser Lys Ile Leu Leu Leu Thr Val Leu Ala Thr
1 5 10 15
Ala Thr Ile Ser Asn Ala Gln Asp Lys Lys Ser Lys Ser Lys Thr Ala
20 25 30
Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu Asn Pro
35 40 45
Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile Leu Asn
50 55 60
Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp Pro Lys
65 70 75 80
Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr Thr Ala
85 90 95
Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu Thr Gly
100 105 110
Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met Arg Gly
115 120 125
Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys Tyr Ser
130 135 140
Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu Gln Arg
145 150 155 160
Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu Lys Val
165 170 175
Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu Lys Asp
180 185 190
Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp Gly Asp
195 200 205
Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr Arg Phe
210 215 220
Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile Leu Asn
225 230 235 240
Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys Trp Gly
245 250 255
Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser Asn Thr
260 265 270
Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val Thr His
275 280 285

Claims (5)

1. An alginate lyase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 3.
2. A DNA molecule encoding the alginate lyase mutant of claim 1.
3. A recombinant expression vector carrying the DNA molecule of claim 2.
4. Bacillus subtilis (B.subtilis)Bacillus subtilis) The Bacillus subtilis comprising the recombinant expression vector of claim 3.
5. Use of the Bacillus subtilis of claim 4 for the production of alginate lyase.
CN201810143677.9A 2018-02-12 2018-02-12 Alginate lyase mutant Active CN110144341B (en)

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Publication number Priority date Publication date Assignee Title
MX2023004260A (en) * 2020-10-29 2023-04-25 Procter & Gamble Cleaning compositions containing alginate lyase enzymes.
CN115725555B (en) * 2022-08-10 2024-10-01 中国海洋大学 Algin lyase mutant
CN115896082B (en) * 2022-08-17 2024-08-06 广西科学院 Alginic acid lyase mutant and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012157814A1 (en) * 2011-05-17 2012-11-22 전남대학교 산학협력단 Metagenome library having alginate lyase activity and novel enzyme alydw
CN102994407A (en) * 2011-12-16 2013-03-27 中国科学院大连化学物理研究所 Flavobacterium strain and incision alginate lyase coding gene, preparation and application
CN109957536A (en) * 2017-12-14 2019-07-02 青岛蔚蓝生物集团有限公司 A kind of bacillus subtilis and its application in alginate lyase production

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WO2012157814A1 (en) * 2011-05-17 2012-11-22 전남대학교 산학협력단 Metagenome library having alginate lyase activity and novel enzyme alydw
CN102994407A (en) * 2011-12-16 2013-03-27 中国科学院大连化学物理研究所 Flavobacterium strain and incision alginate lyase coding gene, preparation and application
CN109957536A (en) * 2017-12-14 2019-07-02 青岛蔚蓝生物集团有限公司 A kind of bacillus subtilis and its application in alginate lyase production

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海藻酸裂解酶异源表达研究进展;赵琳等;《生命科学研究》;20131031(第05期);全文 *

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